Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of art as known to those skilled therein as of the date of the invention described and claimed herein.
Superoxide dismutase (SOD) and the phenomenon of oxygen free radicals (O.sub.2 .sup.-) was discovered in 1968 by McCord and Fridovich (1). Superoxide radicals and other highly reactive oxygen species are produced in every respiring cell as by-products of oxidative metabolism, and have been shown to cause extensive damage to a wide variety of macromolecules and cellular components (for review see 2,3). A group of metalloproteins known as superoxide dismutases catalyze the oxidation-reduction reaction 20.sub.2.sup.- +.sub.2 H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2 and thus provide a defense mechanism against oxygen toxicity. There are three known forms of SODs containing different metals in the protein molecules namely iron, manganese or both copper and zinc. All of them catalyze the same reaction with ultimate efficiency, and all operate by a similar mechanism in which the metal is the catalytic factor in the active site. These enzymes fall into several evolutionary groups. The Mn and Fe-SODs are found primarily in prokaryotic cells while CuZn-SOD have been demonstrated in virtually all eucaryotic organisms (4). Human Cu/Zn SOD-1 is a dimeric metallo-protein composed of identical non-covalently linked subunits, each having a molecular weight of 16000 daltons and containing one atom of copper and one of zinc (5). Each subunit is composed of 153 amino acids whosesequence was established (6,7). Furthermore, a cDNA clone containing the entire coding region of human SOD-1 was recently isolated and sequenced (8,9).
The human Cu-Zn SOD analog produced differs from natural human Cu-Zn SOD in that the amino terminus alanine is not acetylated. The natural human SOD is acetylated at the amino terminus alanine (Hartz, J. W. and Deutsch, H. F., J. Biol. Chem. (1972) 247, 7043-7050, Jabusch, J. R., et al., Biochemistry (1980) 19, 2310-2316; Barra, et al., FEBS Letters (1980) 120, 53 and Oberly, L. W. Superoxide Dismutase, Vol. I, CRC Press, Florida, (1982), pp. 32-33). The natural human SOD is likely to be glycosylated like bovine SOD (Huber, W., U.S. Pat. No. 3,579,495, issued May 18, 1971). Bacterial-produced human SOD is almost certainly not glycosylated as Escherichia coli does not glycosylate proteins which it produces. The amino acid sequence of the bacterial-produced SOD analog is identical to that of mature human SOD and does not contain a methionine residue at its N-terminus.
Since every biological macromolecule can serve as a target for the damaging action of the abundant superoxide radical, interest has evolved in the therapeutic potential of SOD. The scientific literature suggests that SOD could be useful in a wide range of clinical applications. These include prevention of oncogenesis and of tumor promotion and reduction of cytotoxic and cardiotoxic effects of anticancer drugs (10), anti-inflammatory (11), protection of ischemic tissues (12) and protection of spermatozoa (13). In addition, there is great interest in studying the effect of SOD on the aging process (14).
The exploration of the therapeutic potential of human SOD-1 (EC 1.15.1.1) has been limited mainly due to its scarce availability.
To overcome this problem, we have inserted the SOD cDNA of Groner et al. (8) into efficient bacterial expression vectors (copending U.S. patent filed together with this patent). However, although the bacteria produce large amounts of an analog of human Cu/Zn SOD, most of the protein is lacking enzymatic activity.
This invention provides a method for producing in bacteria and purifying an enzymatically active analog of human Cu/Zn SOD.